The present invention relates to the use of extracellular matrix digesting enzymes and neurotoxins to treat various medical conditions/disorders, such as overactive bladder, urinary incontinence due to overactive bladder or unstable detrusor sphincter, benign prostatic hyperplasia and associated bladder voiding complications, urinary retention that is secondary to having a spastic sphincter or a hypertrophied bladder neck, neurogenic bladder dysfunction (e.g. secondary to for example, Parkinson's disease, spinal cord injury, stroke or multiple sclerosis), hyperhidrosis and gall bladder inflammation (cholecystitis).
Neurotoxins, and in particular botulinum toxins, are increasingly finding useful application in treating various medical conditions. Such treatments are typically focally delivered via injections that penetrate the skin or organ lining. This can lead to difficulty in delivering the treatment due to complications from needle penetration, patient concerns such as needle phobia, pain and physician application in the treatment of several urological conditions including overactive bladder (OAB) and detrusor hyperreflexia (DH) which cause bothersome symptoms such as voiding urgency, excessive voiding frequency and incontinence, for example. A detailed discussion of the use and techniques for utilizing botulinum toxin to treat overactive bladder can be found in “Botulinum toxin for the treatment of idiopathic and neurogenic overactive bladder: State of the art” Nitti Victor W. Rev Urol 2006; 8(4):198-208. As detailed therein, botulinum toxin is injected into the bladder wall and the number of injections (between 15 to 50 injections of 100 to 1000 units of botulinum toxin type A and 10 injections of 5000 units botulinum toxin type B) depends on the well known effect and potency difference between the serotype of botulinum toxin utilized, as well as the amount of total toxin and dilution of toxin utilized, as detailed in therein and known in the art.
Incontinence, one symptom of various urologic disorders, includes urge incontinence and stress incontinence. Urge incontinence involves a strong, sudden need to urinate, followed by inappropriate bladder contraction, which then results in leakage. What is troublesome is that it is often the case that these contractions occur regardless of the amount of urine that is in a sufferer's bladder, that is, the bladder does not necessarily have to be so full and under pressure from urine contained therein to result undesirable leakage. Urge incontinence can be a result of neurological injuries (such as spinal cord injury or stroke), neurological diseases (such as multiple sclerosis), infection, bladder cancer, bladder stones, bladder inflammation, or bladder outlet obstruction, for example. While these conditions can be found both in men and women, men have an additional burden in that urge incontinence may also be due to neurologic disease or bladder changes caused by benign prostatic hypertrophy (BPH) or bladder outlet obstruction from an enlarged prostate, for example.
Stress incontinence is an involuntary loss of urine that occurs during physical activity, such as coughing, sneezing, laughing, or exercise. A person can suffer from one or both types of incontinence, and when suffering from both, it is called mixed incontinence. Despite all of the knowledge associated with incontinence, the majority of cases of urge incontinence are idiopathic, which means a specific cause cannot be identified. Urge incontinence may occur in anyone at any age, and it is more common in women and the elderly.
The detrusor of the bladder is the muscle that expels urine from the bladder. Consequences of detrusor hyperreflexia include poor bladder compliance, high intravesical pressure, and reduction in bladder capacity, all of which may result in deterioration of the upper urinary tract.
It is thought that botulinum toxin exerts its effect on bladder hyperactivity by paralyzing the detrusor muscle in the bladder wall or possibly impacting afferent pathways in the bladder and reducing sensory receptors in suburothelial nerves. These effects possibly account for the improvement in urinary incontinence, bladder capacity and reduction in bladder detrusor pressures that are seen when the bladder walls are injected with botulinum toxins. Examples of botulinum toxin use to treat various urologic disorders can be found in “Botulinum Toxin Treatment of Spastic Bladder”, by Dott, C. et al., U.S. Patent App. Publication No. US 2007/0275110A1 and “Methods for the use of neurotoxin in the treatment of urologic disorders”, by Doshi, R., U.S. Patent App. Publication No. 2004/0067235A1, both herein incorporated by reference. Other known potential urological applications for neurotoxins include the treatment of a variety of disorders of the prostate including benign prostatic hyperplasia (BPH), prostatitis, and prostate cancer (see, e.g., U.S. Pat. No. 6,365,164, herein incorporated by reference in its entirety.)
To date, botulinum toxin has shown promising early results for treatment of lower urinary tract symptoms including obstructive and irritative voiding symptoms attributed to BPH. Both subjective (symptoms) and objective (flow rates) improvements have been observed. The prostate is a partially glandular and partially fibromuscular gland of the male reproductive system. During aging, the prostate tends to enlarge (hypertrophy). This prostatic enlargement can lead to urethral obstruction and voiding dysfunction. This is because the urethra passes through the prostate (prostatic urethra) as it leads to the external urethral orifice. A detailed discussion of prostate anatomy (including lobes, stroma, nerve fiber types and innervation) can be found in published U.S. patent application Ser. No. 09/978,982, filed Oct. 15, 2001, and entitled “Use of neurotoxin therapy for treatment of urologic and related disorders”, U.S. Published Patent Application No. 20020025327 A1, herein incorporated by reference in its entirety, in addition to standard anatomy texts.
Botulinum toxin is thought to affect nerve terminals in the prostate and the release of neurotransmitters including acetyicholine, sensory neuropeptides, and noradrenalin. These effects may alter neural control within the prostate. Preliminary reports suggest that botulinum toxin may also have a role in the management of prostate cancer, possibly by inhibiting inflammation and the down regulation of COX-2 expression.
In humans, the gall bladder is the organ that stores about 50 ml of bile (yellow or green alkaline fluid secreted by hepatocytes from the liver of most vertebrates) until needed for digestion. Bile is discharged into the duodenum where it aids the process of digestion of lipids. The gallbladder is about 100 to 120 mm long in humans and is connected to the liver and the duodenum by the biliary tract. A cystic duct connects the gallbladder to the common hepatic duct to form the common bile duct, which then joins the pancreatic duct, and enters through the hepatopancreatic ampulla at the major duodenal papilla. The fundus of the gallbladder is the part farthest from the duct, located by the lower border of the liver at the same level as the transpyloric plane.
Unfortunately, inflammation of the gall bladder, called cholecystitis, can occur and is typically caused by the presence of gall stones (choleliths, crystalline bodies formed by accretion or concretion of normal or abnormal bile components in the gallbladder) which commonly block the cystic duct directly leading to a thickening of the bile, bile stasis, and even secondary infection by gut organisms, predominantly E coli species. This results in inflammation of the wall of the gallbladder. The gallbladder can also become inflamed and infected in the absence of galls stones. This is known as acute acalculous cholecystitis. Chronic, low-level inflammation can lead to a chronic cholecystitis, where the gallbladder is fibrotic and calcified.
In order to gain access and visualize the gall bladder and ducts, endoscopic retrograde cholangiopancreatography (ERCP) can be utilized, a technique that combines the use of endoscopy and fluoroscopy to diagnose and treat problems of the biliary systems. Aided by a video endoscope, ERCP can utilize x-ray examination to investigate and access bile ducts. The inside of the stomach and duodenum can be seen through the endoscope, and dyes are commonly injected by medical personnel into the ducts in the biliary tree so they can be seen on x-rays. ERCP is used primarily to diagnose and treat conditions of the bile ducts, including investigation of and removal gallstones, inflammatory strictures (scars), leaks (from trauma and surgery), and cancer. ERCP combines the use of x-rays and endoscopy and is performed for diagnostic or therapeutic reasons. In some instances, a second camera can be inserted through the channel of the first endoscope (this technique is termed duodenoscope-assisted cholangiopancreatoscopy (DACP) or mother-daughter ERCP). The daughter scope can be used to administer direct electrohydraulic lithotripsy to break up stones, or to help in diagnosis by directly visualizing the duct (as opposed to obtaining X-ray images).
The large size of the botulinum toxin molecule can limit its ability to diffuse, and thus prohibits it from reaching both afferent and efferent nerve fibers. As a result, current methods of administration for OAB, for example, require many injections (typically 20 to 50) of botulinum toxin into the bladder muscle wall or into the prostate. Other examples of botulinum toxin uses includes the treatment of chronic migraine with botulinum toxin, which requires approximately 30 injections into the head and neck musculature, and axillary hyperhidrosis, which requires numerous injections to the dermal skin layer in the axilla (typically anywhere from 10 to 40 injections per axilla, depending on the severity of the condition, area overproducing sweat, size of the patient and concentration, amount and type of botulinum toxin used).
The genus Clostridium has more than one hundred and twenty seven species, grouped according to their morphology and functions. The anaerobic, gram positive bacterium Clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin, which causes a neuroparalytic illness in humans and animals referred to as botulism. The spores of Clostridium botulinum are found in soil and can grow in improperly sterilized and sealed food containers of home based canneries, which are the cause of many of the cases of botulism. The effects of botulism typically appear 18 to 36 hours after eating the foodstuffs infected with a Clostridium botulinum culture or spores. The botulinum toxin can apparently pass unattenuated through the lining of the gut and attack peripheral motor neurons. Symptoms of botulinum toxin intoxication can progress from difficulty walking, swallowing, and speaking to paralysis of the respiratory muscles and death.
About 50 picograms of a commercially available botulinum toxin type A (a purified neurotoxin complex available from Allergan, Inc., of Irvine, Calif. under the trade name BOTOX® in 100 unit vials) is a LD50 in mice (i.e. 1 unit). One unit of BOTOX® contains about 50 picograms (about 56 attomoles) of botulinum toxin type A complex. Interestingly, on a molar basis, botulinum toxin type A is about 1.8 billion times more lethal than diphtheria, about 600 million times more lethal than sodium cyanide, about 30 million times more lethal than cobra toxin and about 1 2 million times more lethal than cholera. Singh, Critical Aspects of Bacteria/Protein Toxins, pages 63-84 (chapter 4) of Natural Toxins II, edited by B. R. Singh et al., Plenum Press, New York (1976) (where the stated LD50 of botulinum toxin type A of 0.3 ng equals 1 unit is corrected for the fact that about 0.05 ng of BOTOX® equals 1 unit). One unit (U) of botulinum toxin is defined as the LD50 upon intraperitoneal injection into female Swiss Webster mice weighing 18 to 20 grams each.
Seven immunologically distinct botulinum neurotoxins have been characterized, these being respectively botulinum neurotoxin serotypes A, B, C1, D, E, F and G, each of which is distinguished by neutralization with type-specific antibodies. The different serotypes of botulinum toxin vary in the animal species that they affect and in the severity and duration of the paralysis they evoke. For example, it has been determined that botulinum toxin type A is 500 times more potent, as measured by the rate of paralysis produced in the rat, than is botulinum toxin type B. Additionally, botulinum toxin type B has been determined to be non-toxic in primates at a dose of 480 U/kg which is about 12 times the primate LD50 for botulinum toxin type A. Moyer E et al., Botulinum Toxin Type 8: Experimental and Clinical Experience, being chapter 6, pages 71-85 of “Therapy With Botulinum Toxin”, edited by Jankovic, J. et al. (1994), Marcel Dekker, Inc. Botulinum toxin apparently binds with high affinity to cholinergic motor neurons, is translocated into the neuron, and blocks the release of acetylcholine. Additional uptake can take place through low affinity receptors, as well as by phagocytosis and pinocytosis.
Regardless of stereotype, the molecular mechanism of toxin intoxication appears to be similar and to involve at least three steps or stages. In the first step of the process, the toxin binds to the presynaptic membrane of the target neuron through a specific interaction between the heavy chain, H chain, and a cell surface receptor; the receptor is thought to be different for each type of botulinum toxin and for tetanus toxin. The carboxyl end segment of the H chain, HC, appears to be important for targeting of the toxin to the cell surface. In the second step, the toxin crosses the plasma membrane of the poisoned cell. The toxin is first engulfed by the cell through receptor-mediated endocytosis, and an endosome containing the toxin is formed. The toxin then escapes the endosome into the cytoplasm of the cell. This step is thought to be mediated by the amino end segment of the H chain, HN, which triggers a conformational change of the toxin in response to a pH of about 5.5 or lower. Endosomes are known to possess a proton pump which decreases intra-endosomal pH. The conformational shift exposes hydrophobic residues in the toxin, which permits the toxin to embed itself in the endosomal membrane. The toxin (or at a minimum the light chain) then translocates through the endosomal membrane into the cytoplasm.
The last step of the mechanism of botulinum toxin activity appears to involve reduction of the disulfide bond joining the heavy chain, H chain, and the light chain, L chain. The entire toxic activity of botulinum and tetanus toxins is contained in the L chain of the holotoxin; the L chain is a zinc (Zn2+) endopeptidase which selectively cleaves proteins essential for recognition and docking of neurotransmitter-containing vesicles with the cytoplasmic surface of the plasma membrane, and fusion of the vesicles with the plasma membrane. Tetanus neurotoxin, botulinum toxin types B, D, F, and G, cause degradation of synaptobrevin (also called vesicle-associated membrane protein (VAMP)), a synaptosomal membrane protein. Most of the VAMP present at the cytoplasmic surface of the synaptic vesicle is removed as a result of any one of these cleavage events. Botulinum toxin serotype A and E cleave SNAP-25. Botulinum toxin serotype C1 was originally thought to cleave syntaxin, but was found to cleave syntaxin and SNAP-25. Each of the botulinum toxins specifically cleaves a different bond, except botulinum toxin type B (and tetanus toxin) which cleave the same bond. Each of these cleavages block the process of vesicle-membrane docking, thereby preventing exocytosis of vesicle content.
Botulinum toxins have been used in clinical settings for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles (i.e. motor disorders). Almost twenty years ago, in 1989, a botulinum toxin type A complex was approved by the U.S. Food and Drug Administration for the treatment of blepharospasm, strabismus and hemifacial spasm. Subsequently, a botulinum toxin type A was also approved by the FDA for the treatment of cervical dystonia and for the treatment of glabellar lines, and a botulinum toxin type B was approved for the treatment of cervical dystonia. Non-type A botulinum toxin serotypes apparently have a lower potency and/or a shorter duration of activity as compared to botulinum toxin type A. Clinical effects of peripheral intramuscular botulinum toxin type A are usually seen within one week of injection. The typical duration of symptomatic relief from a single intramuscular injection of botulinum toxin type A averages about three months, although significantly longer periods of therapeutic activity have been reported.
Although all the botulinum toxin serotypes apparently inhibit release of the neurotransmitter acetylcholine at the neuromuscular junction, they do so by affecting different neurosecretory proteins and/or cleaving these proteins at different sites. For example, botulinum types A and E both cleave the 25 kiloDalton (kD) synaptosomal associated protein (SNAP-25), but they target different amino acid sequences within this protein. Botulinum toxin types B, D, F and G act on vesicle-associated protein (VAMP, also called synaptobrevin), with each serotype cleaving the protein at a different site. Finally, botulinum toxin type C1 has been shown to cleave both syntaxin and SNAP-25. These differences in mechanism of action may affect the relative potency and/or duration of action of the various botulinum toxin serotypes. Apparently, a substrate for a botulinum toxin can be found in a variety of different cell types. See e.g. Biochem J 1;339 (pt 1):159-65.1999, and MovDisord, 10(3):376:1995 (pancreatic islet B cells contains at least SNAP-25 and synaptobrevin).
The molecular weight of the botulinum toxin protein molecule, for all seven of the known botulinum toxin serotypes, is about 150 kD. Interestingly, the botulinum toxins are released by Clostridial bacterium as complexes comprising the 150 kD botulinum toxin protein molecule along with associated non-toxin proteins. Thus, the botulinum toxin type A complex can be produced by Clostridial bacterium as 900 kD, 500 kD and 300 kD forms. Botulinum toxin types B and C1 are apparently produced as only a 700 kD or 500 kD complex. Botulinum toxin type D is produced as both 300 kD and 500 kD complexes. Finally, botulinum toxin types E and F are produced as only approximately 300 kD complexes. The complexes (i.e. molecular weight greater than about 150 kD) are believed to contain a non-toxin hemaglutinin protein and a non-toxin and non-toxic nonhemaglutinin protein. These two non-toxin proteins (which along with the botulinum toxin molecule comprise the relevant neurotoxin complex) may act to provide stability against denaturation to the botulinum toxin molecule, and protection against digestive acids when toxin is ingested. Additionally, it is possible that the larger (greater than about 150 kD molecular weight) botulinum toxin complexes may result in a slower rate of diffusion of the botulinum toxin away from a site of intramuscular injection of a botulinum toxin complex.
In vitro studies have indicated that botulinum toxin inhibits potassium cation induced release of both acetylcholine and norepinephrine from primary cell cultures of brainstem tissue. Additionally, it has been reported that botulinum toxin inhibits the evoked release of both glycine and glutamate in primary cultures of spinal cord neurons and that in brain synaptosome preparations botulinum toxin inhibits the release of each of the neurotransmitters acetylcholine, dopamine, norepinephrine (Habermann E., et al., Tetanus Toxin and Botulinum A and C Neurotoxins Inhibit Noradrenaline Release From Cultured Mouse Brain J Neurochem 51(2);522-527:1988)), CGRP, substance P, and glutamate (Sanchez-Prieto, J., et al., Botulinum Toxin A Blocks Glutamate Exocytosis From Guinea Pig Cerebral Cortical Synaptosomes, Eur J. Biochem 165; 675-681:1897). Thus, when adequate concentrations are used, stimulus-evoked release of most neurotransmitters is blocked by botulinum toxin. See e.g. Pearce, L. B., Pharmacologic Characterization of Botulinum Toxin For Basic Science and Medicine, Toxicon 35(9);1 373-1 412 at 1393; Bigalke H., et al., Botulinum A Neurotoxin Inhibits Non-Cholinergic Synaptic Transmission in Mouse Spinal Cord Neurons in Culture, Brain Research 360;318-324:1985; Habermann E., Inhibition by Tetanus and Botulinum A Toxin of the release of [3H] Noradrenaline and [3H]GABA From Rat Brain Homogenate, Experientia 44;224-226: 1988, Bigalke H., et al., Tetanus Toxin and Botulinum A Toxin Inhibit Release and Uptake of Various Transmitters, as Studied with Particulate Preparations From Rat Brain and Spinal Cord, Naunyn-Schmiedeberg's Arch Pharmacol 31 6;244-251 :1 981, and; Jankovic J. et al., Therapy With Botulinum Toxin, Marcel Dekker, Inc., (1994), page 5.
Botulinum toxin type A can be obtained by establishing and growing cultures of Clostridium botulinum in a fermenter and then harvesting and purifying the fermented mixture in accordance with known procedures. All the botulinum toxin serotypes are initially synthesized as inactive single chain proteins which must be cleaved or nicked by proteases to become neuroactive. The bacterial strains that make botulinum toxin serotypes A and G possess endogenous proteases and serotypes A and G can therefore be recovered from bacterial cultures in predominantly their active form. In contrast, botulinum toxin serotypes C1, D and E are synthesized by nonproteolytic strains and are therefore typically unactivated when recovered from culture. Serotypes B and F are produced by both proteolytic and nonproteolytic strains and therefore can be recovered in either the active or inactive form. However, even the proteolytic strains that produce, for example, the botulinum toxin type B serotype, only cleave a portion of the toxin produced. The exact proportion of nicked to unnicked molecules depends on the length of incubation and the temperature of the culture. Therefore, a certain percentage of any preparation of, for example, the botulinum toxin type B toxin, is likely to be inactive, possibly accounting for the known significantly lower potency of botulinum toxin type B, as compared to botulinum toxin type A (and thus the routine use of many thousands of units of botulinum toxin type B, as known in the art, see e.g. “Long-term safety, efficacy, and dosing of botulinum toxin type B (MYOBLOC®) in cervical dystonia (CD) and other movement disorders” Kumar R and Seeberger L C. Mov Disord 2002; 17(Suppl 5):S292-S293). The presence of inactive botulinum toxin molecules in a clinical preparation will contribute to the overall protein load of the preparation, which has been linked to increased antigenicity, without contributing to its clinical efficacy. Additionally, it is known that botulinum toxin type B has, upon intramuscular injection, a shorter duration of activity and is also less potent than botulinum toxin type A at the same dose level.
High quality crystalline botulinum toxin type A can be produced from the Hall A strain of Clostridium botulinum with characteristics of ≧3×107 U/mg, an A260/A278 of less than 0.60 and a distinct pattern of banding on gel electrophoresis. The known Schantz process can be used to obtain crystalline botulinum toxin type A, as set forth in Schantz, E. J., et al, Properties and use of Botuilnum toxin and Other Microbial Neurotoxins in Medicine, Microbiol Rev. 56;80-99:1992. Generally, the botulinum toxin type A complex can be isolated and purified from an anaerobic fermentation by cultivating Clostridium botulinum type A in a suitable medium. The known process can also be used, upon separation out of the non-toxin proteins, to obtain pure botulinum toxins, such as for example: purified botulinum toxin type A with an approximately 150 kD molecular weight with a specific potency of 1-2×108 LD50 U/mg or greater; purified botulinum toxin type B with an approximately 156 kD molecular weight with a specific potency of 1-2×108 LD50 U/mg or greater; and purified botulinum toxin type F with an approximately 155 kD molecular weight with a specific potency of 1-2×107 LD50 U/mg or greater.
Botulinum toxins and/or botulinum toxin complexes can be obtained from List Biological Laboratories, Inc., Campbell, Calif.; the Centre for Applied Microbiology and Research, Porton Down, U.K.; Wako (Osaka, Japan), Metabiologics (Madison, Wis.) as well as from Sigma Chemicals of St Louis, Mo. Pure botulinum toxin can also be used to prepare a pharmaceutical composition for use in accordance with the present disclosure.
As with enzymes generally, the biological activities of botulinum toxins (which are intracellular peptidases) is dependant, at least in part, upon their 3-dimensional conformation. Thus, botulinum toxin type A is detoxified by heat, various chemicals, surface stretching, and surface drying. Additionally, it is known that dilution of the toxin complex obtained by the known culturing, fermentation and purification to the much lower toxin concentrations used for pharmaceutical composition formulation results in rapid detoxification of the toxin unless a suitable stabilizing agent is present. Dilution of the toxin from milligram quantities to a solution containing nanograms per milliliter presents significant difficulties because of the rapid loss of specific toxicity upon such great dilution. Since the toxin may be used months or years after the toxin containing pharmaceutical composition is formulated, the toxin can be stabilized with a stabilizing agent such as albumin and gelatin.
A commercially available botulinum toxin containing pharmaceutical composition is sold under the trademark BOTOX® (available from Allergan, Inc., of Irvine, Calif.). BOTOX® consists of a purified botulinum toxin type A complex, albumin and sodium chloride packaged in sterile, vacuum-dried form. Botulinum toxin type A is made from a culture of the Hall strain of Clostridium botulinum grown in a medium containing N-Z amine and yeast extract. The botulinum toxin type A complex is purified from the culture solution by a series of acid precipitations to a crystalline complex consisting of the active high molecular weight toxin protein and an associated hemagglutinin protein. The crystalline complex is re-dissolved in a solution containing saline and albumin and sterile filtered (0.2 microns) prior to vacuum-drying. The vacuum-dried product is stored in a freezer at or below −5° C. BOTOX® can be reconstituted with sterile, non-preserved saline prior to intramuscular injection. Each vial of BOTOX® contains about 100 U of Clostridium botulinum toxin type A purified neurotoxin complex, 0.5 milligrams of human serum albumin and 0.9 milligrams of sodium chloride in a sterile, vacuum-dried form without a preservative.
To reconstitute vacuum-dried BOTOX®, sterile normal saline without a preservative (0.9% Sodium Chloride Injection) is used by drawing up the proper amount of diluent in the appropriate size syringe. Since BOTOX® may be denatured by bubbling or similar violent agitation, the diluent is gently injected into the vial. For sterility reasons BOTOX® is preferably administered within four hours after the vial is removed from the freezer and reconstituted. During these four hours, reconstituted BOTOX® can be stored in a refrigerator at about 2° C. to about 8° C. Reconstituted, refrigerated BOTOX® has been reported to retain its potency for at least about two weeks (Neurology, 48:249-53, 1997). It has been reported that botulinum toxin type A has been used in clinical settings as follows:    (1) about 75-125 U of BOTOX® per intramuscular injection (multiple muscles) to treat cervical dystonia;    (2) 5-10 U of BOTOX® per intramuscular injection to treat glabellar lines (brow furrows) (5 units injected intramuscularly into the procerus muscle and 10 units injected intramuscularly into each corrugator supercilii muscle);    (3) about 30-80 U of BOTOX® to treat constipation by intrasphincter injection of the puborectalis muscle;    (4) about 1-5 U per muscle of intramuscularly injected BOTOX® to treat blepharospasm by injecting the lateral pre-tarsal orbicularis oculi muscle of the upper lid and the lateral pre-tarsal orbicularis oculi of the lower lid;    (5) to treat strabismus, extraocular muscles have been injected intramuscularly with between about 1-5 U of BOTOX®, the amount injected varying based upon both the size of the muscle to be injected and the extent of muscle paralysis desired (i.e. amount of diopter correction desired);    (6) to treat upper limb spasticity following stroke by intramuscular injections of BOTOX® into five different upper limb flexor muscles, as follows:    (a) flexor digitorum profundus: 7.5 U to 30 U    (b) flexor digitorum sublimus: 7.5 U to 30 U    (c) flexor carpi ulnaris: 10 U to 40 U    (d) flexor carpi radialis: 15 U to 60 U    (e) biceps brachii: 50 U to 200 U. Each of the five indicated muscles has been injected at the same treatment session, so that the patient receives from 90 U to 360 U of upper limb flexor muscle BOTOX® by intramuscular injection at each treatment session;    (7) to treat migraine, pericranial (injected symmetrically into glabellar, frontalis and temporalis muscles) injection of 25 U of BOTOX® has showed significant benefit as a prophylactic treatment of migraine compared to vehicle as measured by decreased measures of migraine frequency, maximal severity, associated vomiting and acute medication use over the three month period following the 25 U injection.
It is known that botulinum toxin type A can have an efficacy for up to 12 months (European J. Neurology 6 (Supp 4): S111-S1150: 1999), and in some circumstances for as long as 27 months, when used to treat glands, such as in the treatment of hype rhydrosis. See e.g. Bushara K., Botulinum toxin and rhinorrhea, Otolaryngol Head Neck Surg 1996; 114(3):507, and The Laryngoscope 109:1344-1346:1999. However, the usual duration of effect of an intramuscular injection of BOTOX® is typically about 3 to 4 months.
The success of botulinum toxin type A to treat a variety of clinical conditions has led to interest in other botulinum toxin serotypes. Two commercially available botulinum type A preparations for use in humans are BOTOX® available from Allergan, Inc., of Irvine, Calif., and DYSPORT® available from Beaufour Ipsen, Porton Down, England. A botulinum toxin type B preparation (MYOBLOC®) is available from Elan Pharmaceuticals of San Francisco, Calif.
A botulinum toxin has also been proposed for or has been used to treat otitis media of the ear (U.S. Pat. No. 5,766,605), inner ear disorders (U.S. Pat. Nos. 6,265,379; 6,358,926), tension headache, (U.S. Pat. No. 6,458,365), migraine headache pain (U.S. Pat. No. 5,714,468), post-operative pain and visceral pain (U.S. Pat. No. 6,464,986), hair growth and hair retention (U.S. Pat. No. 6,299,893), psoriasis and dermatitis (U.S. Pat. No. 5,670,484), injured muscles (U.S. Pat. No. 6,423,319) various cancers (U.S. Pat. No. 6,139,845), smooth muscle disorders (U.S. Pat. No. 5,437,291), and neurogenic inflammation (U.S. Pat. No. 6,063,768). Controlled release toxin implants are known (see e.g. U.S. Pat. Nos. 6,306,423 and 6,312,708) as is transdermal botulinum toxin administration (U.S. patent application Ser. No. 10/194805).
Additionally, a botulinum toxin may have an effect to reduce induced inflammatory pain in a rat formalin model. Aoki K., et al, Mechanisms of the antinociceptive effect of subcutaneous BOTOX®: Inhibition of peripheral and central nociceptive processing, Cephalalgia September 2003;23(7):649. Furthermore, it has been reported that botulinum toxin nerve blockage can cause a reduction of epidermal thickness. Li Y, et al., Sensory and motor denervation influences epidermal thickness in rat foot glabrous skin, Exp Neurol 1997; 147:452-462 (see page 459). Finally, it is known to administer a botulinum toxin to the foot to treat excessive foot sweating (Katsambas A., et al., Cutaneous diseases of the foot: Unapproved treatments, Clin Dermatol November-December 2002;20(6):689-699; Sevim, S., et al., Botulinum toxin-A therapy for palmar and plantar hyperhidrosis, Acta Neurol Belg December 2002;102(4):167-70), spastic toes (Suputtitada, A., Local botulinum toxin type A injections in the treatment of spastic toes, Am J Phys Med Rehabil October 2002; 81(10):770-5), idiopathic toe walking (Tacks, L., et al., Idiopathic toe walking: Treatment with botulinum toxin A injection, Dev Med Child Neurol 2002;44(Suppl 91):6), and foot dystonia (Rogers J., et al., Injections of botulinum toxin A in foot dystonia, Neurology April 1993;43(4 Suppl 2)). Tetanus toxin, as wells as derivatives (i.e. with a non-native targeting moiety), fragments, hybrids and chimeras thereof can also have therapeutic utility. The tetanus toxin bears many similarities to the botulinum toxins. Thus, both the tetanus toxin and the botulinum toxins are polypeptides made by closely related species of Clostridium (Clostridium tetani and Clostridium botulinum, respectively).
Additionally, both the tetanus toxin and the botulinum toxins are dichain proteins composed of a light chain (molecular weight about 50 kD) covalently bound by a single disulfide bond to a heavy chain (molecular weight about 100 kD). Hence, the molecular weight of tetanus toxin and of each of the seven botulinum toxins (non-complexed) is about 150 kD. Furthermore, for both the tetanus toxin and the botulinum toxins, the light chain bears the domain which exhibits intracellular biological (protease) activity, while the heavy chain comprises the receptor binding (immunogenic) and cell membrane translocational domains.
Additionally, both the tetanus toxin and the botulinum toxins exhibit a high, specific affinity for gangliocide receptors on the surface of presynaptic cholinergic neurons. Receptor mediated endocytosis of tetanus toxin by peripheral cholinergic neurons results in retrograde axonal transport, blocking of the release of inhibitory neurotransmitters from central synapses and a spastic paralysis. Contrarily, receptor mediated endocytosis of botulinum toxin by peripheral cholinergic neurons results in little if any retrograde transport, inhibition of acetylcholine exocytosis from the intoxicated peripheral motor neurons and a flaccid paralysis.
Finally, the tetanus toxin and the botulinum toxins resemble each other in both biosynthesis and molecular architecture. Thus, there is an overall 34% identity between the protein sequences of tetanus toxin and botulinum toxin type A, and a sequence identity as high as 62% for some functional domains. Binz T. et al., The Complete Sequence of Botulinum Neurotoxin Type A and Comparison with Other Clostridial Neurotoxins, J Biological Chemistry 265(16); 9153-9158:1990.
Acetylcholine
Typically only a single type of small molecule neurotransmitter is released by each type of neuron in the mammalian nervous system. The neurotransmitter acetylcholine is secreted by neurons in many areas of the brain, but specifically by the large pyramidal cells of the motor cortex, by several different neurons in the basal ganglia, by the motor neurons that innervate the skeletal muscles, by the preganglionic neurons of the autonomic nervous system (both sympathetic and parasympathetic), by the postganglionic neurons of the parasympathetic nervous system, and by some of the postganglionic neurons of the sympathetic nervous system. Essentially, only the postganglionic sympathetic nerve fibers to the sweat glands, the piloerector muscles and a few blood vessels are cholinergic as most of the postganglionic neurons of the sympathetic nervous system secret the neurotransmitter norepinephine. In most instances acetylcholine has an excitatory effect. However, acetylcholine is known to have inhibitory effects at some of the peripheral parasympathetic nerve endings, such as inhibition of heart rate by the vagal nerve.
The efferent signals of the autonomic nervous system are transmitted to the body through either the sympathetic nervous system or the parasympathetic nervous system. The preganglionic neurons of the sympathetic nervous system extend from preganglionic sympathetic neuron cell bodies located in the intermediolateral horn of the spinal cord. The preganglionic sympathetic nerve fibers, extending from the cell body, synapse with postganglionic neurons located in either a paravertebral sympathetic ganglion or in a prevertebral ganglion. Since, the preganglionic neurons of both the sympathetic and parasympathetic nervous system are cholinergic, application of acetylcholine to the ganglia will excite both sympathetic and parasympathetic postganglionic neurons.
Acetylcholine activates two types of receptors, muscarinic and nicotinic receptors. The muscarinic receptors are found in all effector cells stimulated by the postganglionic neurons of the parasympathetic nervous system, as well as in those stimulated by the postganglionic cholinergic neurons of the sympathetic nervous system. The nicotinic receptors are found in the synapses between the preganglionic and postganglionic neurons of both the sympathetic and parasympathetic. The nicotinic receptors are also present in many membranes of skeletal muscle fibers at the neuromuscular junction.
Acetylcholine is released from cholinergic neurons when small, clear, intracellular vesicles fuse with the presynaptic neuronal cell membrane. A wide variety of non-neuronal secretory cells, such as, adrenal medulla (as well as the PC12 cell line) and pancreatic islet cells release catecholamines and parathyroid hormone, respectively, from large dense-core vesicles. The PC12 cell line is a clone of rat pheochromocytoma cells extensively used as a tissue culture model for studies of sympathoadrenal development. Botulinum toxin inhibits the release of both types of compounds from both types of cells in vitro, permeabilized (as by electroporation) or by direct injection of the toxin into the denervated cell. Botulinum toxin is also known to block release of the neurotransmitter glutamate from cortical synaptosomes cell cultures.
A variety of substances, termed proteolytic enzymes, degrade or digest substances found in the extracellular matrix. These include the family of hyaluronidases, plasminogen activators and collagenase, for example. Hyaluronidase causes hydrolysis of hyaluronic acid, a polysaccharide (nonsulfated glycosaminoglycan) found in the intercellular matrix of connective tissue. Hyaluronidase temporarily reduces the viscosity of the extracellular matrix (tissue cement) by digesting hyaluronic acid or hyaluronate, which is widely distributed throughout connective, epithelial, and neural tissues. This effect promotes the diffusion or spread of other drugs like anesthetic agents. Hyaluronidase may be injected into connective tissue to enhance the effects of co-injected drugs.
Hyaluroronidase can be obtained from a variety of sources and is typically derived from testicular tissue extracts. For example, ISTA Pharmaceuticals of Irvine, Calif., USA manufactures and distributes VITRASE (a sheep sourced (ovine) form of hyaluronidase), which is just one example of a hyaluronidase for injection. VITRASE is an injectable drug approved by the U.S. FDA as an adjunct to (in combination with) other injected drugs to increase their absorption and dispersion. As stated previously, hyaluronidase has been used most commonly in combination with local anesthetics in the setting of ophthalmic (eye) surgery. Hyaluronidase increases tissue permeability and promotes the spread or dispersion of other drugs, for example, speeding the onset of action for an anesthetic. VITRASE is also approved for use as an adjunct to rehydrating agents, and for use with certain imaging agents. Hyaluronidase is also available as a recombinant purified preparation of the enzyme recombinant human hyaluronidase, an example of which is HYLENEX, which is marketed by Baxter Healthcare Corporation, Deerfield, Ill., USA. HYLENEX (a recombinant hyaluronidase) is available as a sterile clear, colorless, nonpreserved ready for use solution (each mL containing 150 USP units of recombinant human hyaluronidase with 8.f mg sodium chloride, 1.4 mg bibasic sodium phosphate, 1.0 mg human albumin, 0.9 mg edetate, 0.3 mg calcium chloride, and sodium hydroxide for pH adjustment. Another exemplary hyaluronidase produced from sheep testes is named HYALASE, by Aventis Pharma, Lane Cove, NSW, Australia.
Hyaluronidase increases dispersion in the interstitial matrix provided local pressure is adequate to furnish the necessary mechanical impulse. Such an impulse is normally initiated by injected solutions and the rate of diffusion is proportionate to the amount of enzyme. The extent of diffusion is also proportionate to the volume of solution, as known in the art.
Investigation of maintenance of efficacy, spread of effect and decrease in required dose of botulinum toxin administered along with hyaluronidase for treating axillary hyperhidrosis has been reported (“Diffusion and short-term efficacy of botulinum toxin A after the addition of hyaluronidase and its possible application for the treatment of axillary hyperhidrosis” by Goodman G. Dermatol Surg May 2003; 29(5):533-8. Here a formulation/mixture containing a botulinum toxin and a hyaluronidase is injected to treat hyperhidrosis, as well as administration of botulinum toxin and superadded hyaluronidase.
Other proteolytic enzymes include collagenase and plasminogen activators which digest extracellular matrix proteins. Plasminogen activators (PA) belong to a class of serine proteases that have considerable substrate specificity and convert the inactive zymogen plasminogen to plasmin. Plasmin is a general protease which is capable of degrading many proteins including laminin, fibronectin and activating latent collagenase moieties.
What is needed therefore is a method for treating various disorders that reduces the amount of botulinum toxin administered to a patient. More particularly a method is needed that reduces, or more preferably even eliminates, the number of, or need for, injection of neurotoxins, such as botulinum toxins, to treat various disorders.